There is considerable evidence in the literature that deaths due to the simultaneous abuse of ethanol with opioids such as heroin or prescription opioids leads to lower blood levels of the opioid upon autopsy than is observed following death due to the opioid alone. We have carried out an extensive study which has shown that either ethanol or diazepam at doses that do not alter the acute effects of morphine reversed the antinociceptive tolerance produced by chronic morphine. The mechanism of the reversal produced by the two drugs differed in that inhibitors of either GABAA or GABAB receptors alone did not reverse the effect of alcohol but blocking both type GABA receptors completely reversed the effect of ethanol on morphine tolerance. Conversely the inhibition of morphine tolerance produced by diazepam was completely reversed by a GABAA inhibitor alone but not by the GABAB inhibitor. The overall objective of the work described in this proposal is to elucidate the cellular mechanisms by which opioid tolerance is reversed by the concomitant administration of ethanol and other agents affecting the central nervous system. Using genetically altered mice and a number of molecular techniques we will elucidate further the role of GABA receptors and the signaling mechanisms involved in these effects. In the second specific aim we propose to elucidate whether ethanol and diazepam reverses the tolerance to opioids such as buprenorphine and methadone which are used in maintenance therapy and oxycodone and other prescription opioids with a history of abuse. We and others have reported differences in the mechanism of tolerance development for various opioids. For instance, protein kinase A and protein kinase C inhibitors but not GIRK inhibitors reversed tolerance to morphine and other moderately efficacious opioids but not to the high efficacy DAMGO. GIRK inhibitors revered the tolerance to DAMGO but not the other opioids. These differences in the mechanism of tolerance development to various opioids cause us to investigate whether ethanol and/or diazepam will reverse the tolerance to opioids of various efficacies. Obviously, we will elucidate the differing mechanisms if the opioids are affected differently by ethanol or diazepam. In the third specific aim we will investigate whether ethanol and diazepam also reverse tolerance to a peripheral effect of opioids. We and others have found certain differences between the mechanisms of opioid tolerance in the brain and the gastrointestinal tract and now we propose to determine whether ethanol and/or diazepam reverses opioid tolerance in the gastrointestinal tract. The information gained from these experiments will provide important information as we and others continue to elucidate the mechanism of opioid tolerance.